Espirito Santo virus (ESV) is a newly discovered virus recovered as contamination in a sample of a virulent strain of dengue-2 virus (strain 44/2), which was recovered from a patient in the state of Espirito Santo, Brazil, and amplified in insect cells. ESV was found to be dependent upon coinfection with a virulent strain of dengue-2 virus and to replicate in C6/36 insect cells but not in mammalian Vero cells. A sequence of the genome has been produced by de novo assembly and was not found to match to any known viral sequence. An incomplete match to the nucleotide sequence of the RNA-dependent RNA polymerase from Drosophila X virus (DXV), another birnavirus, could be detected. Mass spectrometry analysis of ESV proteins found no matches in the protein data banks. However, peptides recovered by mass spectrometry corresponded to the de novo-assembled sequence by BLAST analysis. The composition and three-dimensional structure of ESV are presented, and its sequence is compared to those of other members of the birnavirus family. Although the virus was found to belong to the family Birnaviridae, biochemical and sequence information for ESV differed from that of DXV, the representative species of the genus Entomobirnavirus. Thus, significant differences underscore the uniqueness of this infectious agent, and its relationship to the coinfecting virus is discussed.
The immunogenicity and safety of three novel host-range vaccines containing deletions in the transmembrane domain of dengue virus serotype 2 (DV2) E glycoprotein were evaluated in African green monkeys. The shorter transmembrane domains are capable of functionally spanning an insect but not a mammalian cell membrane, resulting in production of viral mutants that have reduced infectivity in mammalian hosts but efficient growth in insect cells. Groups of four monkeys received one dose each of test vaccine candidate with no booster immunization. After immunization, levels of viremia produced by each vaccine were determined by infectious center assay. Vaccine recipient immune response to wild-type DV2 challenge was measured on Day 57 by enzyme-linked immunosorbent assay and plaque reduction neutralization test. Two vaccines, DV2ΔGVII and DV2G460P, generated neutralizing antibody in the range of 700–900 50% plaque reduction neutralization test units. All three vaccine strains decreased the length of viremia by at least two days. No safety concerns were identified.
BackgroundThere are over 700 known arboviruses and at least 80 immunologically distinct types that cause disease in humans. Arboviruses are transmitted among vertebrates by biting insects, chiefly mosquitoes and ticks. These viruses are widely distributed throughout the world, depending on the presence of appropriate hosts (birds, horses, domestic animals, humans) and vectors. Mosquito-borne arboviruses present some of the most important examples of emerging and resurgent diseases of global significance.MethodsA strategy has been developed by which host-range mutants of Dengue virus can be constructed by generating deletions in the transmembrane domain (TMD) of the E glycoprotein. The host-range mutants produced and selected favored growth in the insect hosts. Mouse trials were conducted to determine if these mutants could initiate an immune response in an in vivo system.ResultsThe DV2 E protein TMD defined as amino acids 452SWTMKILIGVIITWIG467 was found to contain specific residues which were required for the production of this host-range phenotype. Deletion mutants were found to be stable in vitro for 4 sequential passages in both host cell lines. The host-range mutants elicited neutralizing antibody above that seen for wild-type virus in mice and warrant further testing in primates as potential vaccine candidates.ConclusionsNovel host-range mutants of DV2 were created that have preferential growth in insect cells and impaired infectivity in mammalian cells. This method for creating live, attenuated viral mutants that generate safe and effective immunity may be applied to many other insect-borne viral diseases for which no current effective therapies exist.
A vaccine against Chikungunya virus (ChikV), a reemerging pathogenic arbovirus, has been made by attenuating wild-type (WT) virus via truncation of the transmembrane domain (TMD) of E2 and selecting for host range (HR) mutants. Mice are a standard model system for ChikV disease and display the same symptoms of the disease seen in humans. Groups of mice were inoculated with one of three ChikV HR mutants to determine the ability of each mutant strain to elicit neutralizing antibody and protective immunity upon virus challenge. One mutant, ChikV TM17-2, fulfilled the criteria for a good vaccine candidate. It displayed no reactogenicity at the site of injection, no tissue disease in the foot/ankle and quadriceps, and no evidence of viral persistence in foot/ankle tissues 21 days after infection. Upon challenge with a highly pathogenic strain of ChikV, the mutant blocked viral replication in all tissues tested. This study identified a ChikV HR mutant that grows to high levels in insect cells but was restricted in the ability to assemble virus in mammalian cells in vitro. The study demonstrates that these HR strains are attenuated in the mammalian host and warrant further development as live-attenuated vaccine strains.
Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.
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